Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

Gavin, James M.; Hoar, Kara M.; Xu, Qing; Ma, Jingya; Lin, Yu-Hua; Chen, Jiejin; Chen, Wei; Bruzzese, Frank J.; Harrison, Sean; Mallender, William D.; Bump, Nancy J.; Sintchak, Michael D.; Bence, Neil; Li, Ping; Dick, Lawrence R.; Gould, Alexandra E.; Chen, Jesse J.

doi:10.1074/jbc.m114.573972

Cited by 36 publications

(32 citation statements)

References 42 publications

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“…That, therefore, suggests that binding of UFC1, similar to UFM1, can stabilize the dimeric UBA5 and thereby assist in ATP binding. Indeed, previously, Gavin et al (32) demonstrated that UFC1 stimulates the formation of the UBA5 to UFM1 thioester.…”

Section: Discussionmentioning

confidence: 92%

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

Mashahreh¹,

Hassouna²,

Soudah³

et al. 2018

FASEB j.

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All ubiquitin-like proteins (UBLs) undergo an activation process before their conjugation to target proteins. Although the steps required for the activation of UBLs are conserved and common to all UBLs, we have previously shown that the activation of the UBL, ubiquitin fold modifier 1 (UFM1) by the E1, Ufm1 modifier-activating enzyme 5 (UBA5) is executed in a trans-binding mechanism, not observed in any other E1. In this study, we explored the necessity of that mechanism for UFM1 activation and found that it is needed not only for UFM1 binding to UBA5 but also for stabilizing the UBA5 homodimer. Although UBA5 functions as a dimer, in solution it behaves as a weak dimer. Dimerization of UBA5 is required for ATP binding; therefore, stabilization of the dimer by UFM1 enhances ATP binding. Our results make a connection between the binding of UFM1 to UBA5 and the latter's affinity to ATP, so we propose a novel mechanism for the regulation of ATP's binding to E1.-Mashahreh, B., Hassouna, F., Soudah, N., Cohen-Kfir, E., Strulovich, R., Haitin, Y., Wiener, R. Trans-binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding.

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Section: Discussionmentioning

confidence: 92%

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

Mashahreh¹,

Hassouna²,

Soudah³

et al. 2018

FASEB j.

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“… 89 , 139 This would facilitate the formation of an E1∼UFM1 thioester, a process that is also stimulated by binding of E2 UFC1 . 141 Contrary to canonical E1s, UBA5 does not undergo a second round of adenylation following thioester formation. 141 However, thioester transfer is accelerated by E1 UBA5 binding of ATP and magnesium.…”

Section: E1 Activating Enzymesmentioning

confidence: 99%

“… 141 Contrary to canonical E1s, UBA5 does not undergo a second round of adenylation following thioester formation. 141 However, thioester transfer is accelerated by E1 UBA5 binding of ATP and magnesium. 141 Similar to E1 ATG7 , thioester transfer of UFM1 from E1 UBA5 to E2 UFC1 was shown to occur via a trans mechanism.…”

Section: E1 Activating Enzymesmentioning

confidence: 99%

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism

2017

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Ubiquitin-like proteins (Ubl’s) are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions. Similar to ubiquitin, conjugation is achieved through a cascade of activities that are catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. In this review, we will summarize structural and mechanistic details of enzymes and protein cofactors that participate in Ubl conjugation cascades. Precisely, we will focus on conjugation machinery in the SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 pathways while referring to the ubiquitin pathway to highlight common or contrasting themes. We will also review various strategies used to trap intermediates during Ubl activation and conjugation.

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“…Although the only step in UFM1 conjugation that requires energy is UFM1 activation by UBA5, ATP also plays a role in the transfer of UFM1 from UBA5 to UFC1. Specifically, Gavin et al have shown that charged UBA5 (UBA5~UFM1) hardly transfers UFM1 to UFC1 if ATP is missing [37]. In that case hydrolysis of ATP is not required but only binding of ATP to the UBA5~UFM1 adduct.…”

Section: The N-terminus Of Uba5 Contributes To the Transfer Of Ufm1 Fmentioning

confidence: 99%

An N-Terminal Extension to UBA5 Adenylation Domain Boosts UFM1 Activation: Isoform-Specific Differences in Ubiquitin-like Protein Activation

Soudah¹,

Padala²,

Hassouna³

et al. 2019

Journal of Molecular Biology

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Modification of proteins by the ubiquitin-like protein, UFM1, requires activation of UFM1 by the E1-activating enzyme, UBA5. In humans UBA5 possesses two isoforms, each comprising an adenylation domain, but only one containing an N-terminal extension.Currently the role of the N-terminal extension in UFM1 activation is not clear. Here we provide structural and biochemical data on UBA5 N-terminal extension to understand its contribution to UFM1 activation. The crystal structures of the UBA5 long isoform bound to ATP with and without UFM1 show that the N-terminus not only is directly involved in ATP binding, but also affects how the adenylation domain interacts with ATP.Surprisingly, in the presence of the N-terminus, UBA5 no longer retains the 1:2 ratio of ATP to UBA5, but rather this becomes a 1:1 ratio. Accordingly, the N-terminus significantly increases the affinity of ATP to UBA5. Finally, the N-terminus, although not directly involved in the E2 binding, stimulates transfer of UFM1 from UBA5 to the E2, UFC1.

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Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

Cited by 36 publications

References 42 publications

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism

An N-Terminal Extension to UBA5 Adenylation Domain Boosts UFM1 Activation: Isoform-Specific Differences in Ubiquitin-like Protein Activation

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